1. Field of the Invention
The present invention relates to a method for determining the ability of a compound to modify the interaction between parkin and the p38 protein, and in particular a method for screening for or detecting compounds intended for the prevention and/or treatment of neurodegenerative pathological conditions.
It also relates to compounds intended for the prevention and/or treatment of neurodegenerative pathological conditions which may modify the interaction between parkin and the p38 protein.
2. Description of the Related Art
The parkin gene is mutated in certain familial forms (autosomal recessive juvenile forms) of Parkinson's disease (Kitada et al., 1998). Parkinson's disease (Lewy, 1912) is one of the most common neurodegenerative diseases, affecting more than 1% of the population over 55 years of age. Patients suffering from this disease have neurological disorders which are grouped together under the term Parkinson's Syndrome, characterized by a rigidity, bradykinesia, and shaking while resting. These symptoms are the consequence of a degeneration of the dopaminergic neurons of the substantia nigra of the brain.
Most patients suffering from Parkinson's disease do not have a family history. However, some of the familial cases correspond to a monogenic form of the disease. At the present time, only three different genes have been identified in certain rare hereditary forms. The first form corresponds to an autosomal dominant form, in which the gene responsible encodes alpha synuclein (Polymeropoulos et al., 1997). This protein is an abundant constituent of the intracytoplasmic inclusions called Lewy bodies, which are used as a marker for Parkinson's disease (Lewy, 1912). The second form, also autosomal dominant, is associated with a mutation in a gene encoding a hydrolase called ubiquitin carboxy-terminal hydrolase Li (Leroy et al., 1998). This enzyme is presumed to hydrolyse ubiquitin polymers or conjugates to ubiquitin monomers. The third form differs from the previous forms in that it has an autosomal recessive transmission and often begins before 40 years of age, and also in that there is an absence of Lewy bodies. These diseases respond more favourably to levodopa, a dopamine precursor which is used as treatment for Parkinson's disease. The gene involved in this form encodes a novel protein called parkin (Kitada et al., 1998).
The parkin gene consists of 12 exons which cover a genomic region of more than 500 000 base pairs on chromosome 6 (6q25.2-q27). At the present time, two major types of mutation of this gene, which are the cause of the disease, are known, either deletions of varying size in the region which covers exons 2 to 9, or point mutations which produce the premature appearance of a stop codon or a change of amino acid (Kitada et al., 1998; Abbas et al., 1999; Lucking et al., 1998; Hattori et al., 1998). The nature of these mutations and the autosomal recessive method of transmission suggests a loss of function of parkin, leading to Parkinson's disease.
This gene is expressed in a large number of tissues, and in particular in the substantia nigra. Several transcripts corresponding to this gene exist, which originate from different alternative splicings (Kitada et al., 1998; Sunada et al., 1998). In the brain, two types of messenger RNA are found, of which one lacks the portion corresponding to exon 5. In leukocytes, parkin messenger RNAs which do not contain the region encoding exons 3, 4 and 5 have been identified. The longest of the parkin messenger RNAs, which is present in the brain, contains 2 960 bases and encodes a 465 amino acid protein.
This protein has weak homology, in its N-terminal portion, with ubiquitin. Its C-terminal half contains two ring finger motifs, separated by an IBR (In Between Ring) domain, corresponding to a cysteine-rich region able to bind metals, like the zinc finger domains (Morett, 1999). It has been shown, by immunocytochemistry, that parkin is located in the cytoplasm and the Golgi apparatus of neurons of the substantia nigra which contain melanin (Shimura et al., 1999). In addition, this protein is present in certain Lewy bodies of Parkinsonians. Recent studies indicate that parkin functions like E3 ubiquitin-protein ligase: an enzyme which is thought to facilitate the transfer of ubiquitin from a protein called E2 ubiquitin-conjugating enzyme to target proteins called upon to be degraded by a proteasome-dependent process (Shimura et al., 2000). One hypothesis would be that parkin has a protective role against the accumulation of incorrectly conformed proteins coming from the endoplasmic reticulum which, if not degraded, would induce a neurotoxic stress leading to neuronal death (Imai et al., 2000). In the autosomal recessive juvenile forms, parkin is absent, thus confirming that the loss of this function is responsible for the disease.
Elucidation of the exact role of the parkin protein in the process of degeneration of dopaminergic neurons is therefore determinant for the understanding of and the therapeutic approach to Parkinson's disease, and more generally diseases of the central nervous system.
In addition, in terms of the discovery of novel molecules for treating Parkinson's disease, and in general diverse neurodegenerative diseases, the stakes are high for public health. This is because there is, however, no specific screening method for such molecules which can be used at high throughput.